A cholesterol-dependent endocytic mechanism generates midbody tubules during cytokinesis

Electron tomography reveals that many vesicles within the intracellular bridge (ICB) of a cytokinetic cell are not independent but connected, forming a newly described ICB vesicular structure - narrow midbody tubules that are often branched. Endocytosis occurs at the rim of the ICB via a dynamin-independent cholesterol-dependent mechanism and contributes to the extensive accumulation of membrane-bound structures in the ICB. Thus, ICB vesicles generated via endocytosis are not solely dependent on the recycling endocytic pathway but also involve locally occurring endocytosis. Red, plasma membrane; green, microtubules; dark blue, small vesicles; yellow, tubules within the ICB; white, small vesicles and tubules within peri-ICB; orange, multivesicular bodies; light blue, membrane which is unclear if it is continuous with outer plasma membrane. Cytokinesis is the final stage of cell division and produces two independent daughter cells. Vesicles derived from internal membrane stores, such as the Golgi, lysosomes, and early and recycling endosomes accumulate at the intracellular bridge (ICB) during cytokinesis. Here, we use electron tomography to show that many ICB vesicles are not independent but connected, forming a newly described ICB vesicular structure - narrow tubules that are often branched. These 'midbody tubules' labelled with horseradish peroxidase (HRP) within 10min after addition to the surrounding medium demonstrating that they are derived from endocytosis. HRP-labelled vesicles and tubules were observed at the rim of the ICB after only 1min, suggesting that midbody tubules are likely to be generated by local endocytosis occurring at the ICB rim. Indeed, at least one tubule was open to the extracellular space, indicative of a local origin within the ICB. Inhibition of cholesterol-dependent endocytosis by exposure to methyl-β-cyclodextrin and filipin reduced formation of HRP-labelled midbody tubules, and induced multinucleation following ICB formation. In contrast, dynamin inhibitors, which block clathrin-mediated endocytosis, induced multinucleation but had no effect on the formation of HRP-labelled midbody tubules. Therefore, our data reveal the existence of a cholesterol-dependent endocytic pathway occurring locally at the ICB, which contributes to the accumulation of vesicles and tubules that contribute to the completion of cytokinesis.